基于FXR-NLRP3通路探讨京尼平甘酸抗胆汁淤积肝炎机制的研究

We previously showed that geniposidic acid (GPA), a chemical from oldenlandia diffusa acts as rat hepatic Fxr inducer and upregulates expression of Bsep/Mrp2 at mRNA and protein level, thus leading to improve ANIT-induced cholestatic liver injury. However, the mechanism how GPA plays a hepatoprotective role is largely unknown. In the present study, we are planning to explore whether FXR activation can inhibit LPS-induced NLRP3 expression, a gen form NOD like receptor family 3 (NLRP3) assembling with ASC and pro-caspase-1 as NLRP3 inflammasome and NLRP3 promoter activity. Next, we also investigate whether FXR can bind to the FXRE at the NLRP3 promoter region containing 2 core binding sites of NF-кB, consequently resulting to inhibit NLRP3 expression at transcriptional level. Besides, we also study whether FXR activation can suppress the transloction of NF-кB. Furthermore, we will further study whether FXR activation can block activation of NLRP3 inflammasome, causing to reduce IL-1β/18 production. Subsequently, Fxr-/- mice are used to study whether Fxr activation can inhibit Nlrp3 expression in vivo. According to the following-mentioned studies, we will demonstrate that NLRP3 is a target gene of FXR, and FXR-NLRP3 axis is essential for prevention/treatment of cholestasis. At last, we will aim to study whether GPA improves ANIT-induced liver injury and inhibits inflammation by regulation of FXR-NLRP3 axis in mice.

我们前期研究已阐述了白花蛇舌草成分京尼平甘酸诱导FXR的表达，促进胆汁酸排泄，改善ANIT诱导的胆汁淤积肝炎，但作用机制有待研究。本项目拟通过研究FXR活化对LPS诱导的NLRP3表达及其启动子活性、NF-кB转位、NLRP3炎症小体活化和IL-1β/IL-18成熟与分泌的影响，找寻FXR与NLRP3启动子FXRE的结合位点，以及研究肝内-肝外胆汁淤积动物肝组织中Fxr与Nlrp3等基因的表达，探讨NLRP3是FXR的靶基因，阐述FXR在转录水平上调控NLRP3表达，导致抑制胆汁淤积肝炎，为FXR-NLRP3路径成为胆汁淤积防治的新靶点提供理论依据。最后，基于FXR-NLRP3通路，通过体内-体外实验，研究京尼平甘酸激活FXR抑制NLRP3的表达，降低炎症，改善ANIT/BDL诱导的肝内/肝外胆汁淤积肝炎，从而阐述京尼平甘酸通过调控FXR-NLRP3路径是其发挥抗胆汁淤积肝炎的机制。

白花蛇舌草功效“清热利湿”，具有保肝、利胆、抗炎作用，但机制不明。本研究，首先，论证FXR调控NLRP3炎症小体表达的机制，进而揭示NLRP3是FXR的靶基因，阐述FXR通过竞争NF-κB在NLRP3启动子结合位点，在转录水平上抑制NLRP3表达的机制，为FXR-NLRP3通路防治胆汁淤积肝病提供了新的理论依据。并在此基础上，发现并解析了SIRT6通过去乙酰化作用调控FXR的表达及其转录活性的关键机制，为胆汁淤积肝病的防治提供了新靶点。其次，以京尼平苷酸GPA作为白花蛇舌草代表成分，我们首次发现，并论证了GPA是一个新颖的FXR配体激动剂和共价NLRP3抑制剂，并基于FXR-NLRP3通路在防治胆汁淤积肝病作用的重要性，重点解析了GPA通过调控FXR和NLRP3两个胆汁淤积肝病发病的关键靶点，降低原代肝细胞和库否细胞之间炎症信号的交谈，改善肝脏炎症，并逆转胆汁淤积肝病的胆汁酸合成、转运、代谢紊乱，发挥保肝、抗炎、抗胆汁淤积肝病的机制，为白花蛇舌草及其成分京尼平苷酸GPA的新药开发和临床应用奠定了基础。同时，也为“清热利湿”中医药防治胆汁淤积肝病提供了新理论和实验依据。